The invention pertains to a universal method for producing highly-active biologically active compounds in general, and, more particularly, to do so by immobilization with a covalent linkage on macroporous polymeric carriers in the presence of higher concentrations of inorganic salts.
Research and application of immobilized, biologically active compounds represents today an extensive field involving both development and application projects of considerable practical importance.
The preparation and application of insoluble enzymes by bonding a soluble enzyme to a carrier enables one to use the catalyst system repeatedly in a batch or through-flow arrangement of reaction in which the insoluble enzyme has the role of the specific heterogeneous catalyst. Such arrangement of the catalytic enzymatic reaction facilitates the subsequent separation of the enzyme from substrate and reaction products, and lowers the cost while enabling one to automate the process in multiple or continuous performance.
The rate of enzymatically catalyzed reaction, insofar as the interaction of a substrate with the covalently immobilized enzyme, depends also on the character of linkage enzyme-carrier, i.e. on physical and chemical properties of the carrier (the size and distribution of pores, hydrophilicity of surface, etc.). Hydrodynamic parameters of the system, the flow rate of substrate, temperature, and others also significantly influence the final yield of reaction. The detailed knowledge of the factors affecting enzymatically catalyzed reactions utilizing the immobilized enzymes was obtained also by investigations of the processes on a preparative scale (penicilinacylase) (I. CHIBATA, Immobilized Enzymes, J. Wiley and sons, New York 1970).
Immobilized, biologically active compounds have found a very broad application in affinity chromatography. This method employs the capacity of numerous biologically active compounds to form sorption complexes with other compounds, while the character of interaction is very specific and reversible. If one of the components of a sorption complex is bonded to a solid carrier, only such compounds that are noted for their specific affinity to the bonded compound are selectively adsorbed from solution under suitable conditions. The sorption complex may be easily separated from other components in solution by means of the carrier. Dissociation of the sorption complex and separation of the soluble component from the component which remains chemically bonded to the carrier occur by change of conditions (pH, ionic strength, temperature, addition of competitive sorbates, and the like). This procedure finds use in the isolation and purification of enzymes, enzyme inhibitors, antidotes, antigens, soluble proteins, etc. Immobilization of bioactive compounds is advantageously used in biochemical analysis in the application of radioactive-labelled compounds or in optical labelling.
Numerous materials are employed as carriers of biologically active compounds, for example, porous glass, silica gel, activated carbon, cellulose and its derivatives, starch, agarose, cross-linked polydextrans, synthetic polymers and copolymers such as polyacrylamide, polystyrene, polyamides, poly(maleic anhydride), polyacrylates, polymethacrylates, etc. Some types are unsuitable because they carry ionogenic functional groups; others are noted for their strong non-specific sorption properties for proteins. Other types have insufficient mechanical, hydrolytical, microbial or thermal stability or unsuitable pore size distribution. These features considerably narrow their region of application. Homogeneous hydrophilic carriers of polysaccharide type mainly must not dry out, a fact which makes their storage and transportation difficult.
Most of the above-mentioned shortcomings are overcome in carriers prepared by copolymerization of 2-hydroxylalkyl methacrylates with alkylene dimethacrylates, which are activated by substitution on the hydroxyl group for the purpose of immobilization reaction (J. Coupek, J. Turkova, O. Hubalkova, M. Krivakova; Czechoslovak Pat. No. 167,530).
Further detailed studies concerning the mechanisms of immobilization of biologically active compounds reveal that the covalent linkage has to be as stable as possible towards hydrolysis, which cannot be satisfied, for example, by activation with cyanogen bromide or by amide, sulfide, ester or other bonding groups. Immobilization proved most suitable utilizing reactive epoxide groups according to the following scheme: ##STR1## where Prot means the residue of protein.
Experiments with immobilization on the copolymer of glycidyl methacrylate with ethylene dimethacrylate (J. Turkova, K. Blaha, M. Malanikova, D. Vajenerova, F. Svec and J. Kalal; Biochem. Biophys. Acts 5424 (1978) (62) revealed that the reactive glycidyl methacrylate groups enclosed in a strongly cross-linked matrix of copolymer cannot be wholly employed for immobilization, and that their subsequent deactivation is difficult. Therefore, a macroporous copolymer of 2-hydroxyethyl methacrylate with ethylene dimethacrylate was advantageously used as a carrier which was activated with epichlorohydrine and contained epoxypropyl functional groups only on the inner surfaces of porous particles (J. Turkova, K., J. Horacek, J. Vagener, A. Frydrychove and J. Coupek; J. Chromatogr. 215 (1981) (165-179) and on the surface of particles.
However, the yield of immobilized activity was rather low in some cases of direct bonding of protein on the epoxypropyl derivative of the hydroxyethyl methacrylate copolymer. This shortcoming, which occurs also in several other known activations of synthetic polymeric carriers, is overcome by the subject of this invention.